1 during each round of cell division, the successful partitioning of chromosomes is carried out by the mitotic spindle, a chemomechanical machine that is comprised of microtubules, molecular motors, and associated regulatory proteins. Due to its central role in cell biology and because it is a target for antimitotic cancer therapies, there is a large research effort underway to understand mechanisms underlying spindle morphogenesis and maintenance. Experiments in cells have defined a number of molecular motors and other proteins that are involved in mitosis. However, because of the large number of molecules involved and the built-in functional redundancy employed to prevent chromosome missegregation, there are many unresolved questions regarding the precise molecular interactions and physical mechanisms driving mitosis. In particular, there is a gap between single-molecule investigations on isolated proteins and the behavior observed in the complex environment of the cell. The goal of this project is to assemble microtubules into artificial mitotic spindles in vitro, and to apply this novel experimental tool to study the performance of specific motor and microtubule binding proteins in a geometry resembling that found in dividing cells. The artificial spindles will be assembled using microfabricated chambers, AC electric fields and surface patterned motor proteins, and the resulting structures will be characterized using fluorescence microscopy. Specific experiments will be carried out to test the sufficiency of current models of spindle morphogenesis and maintenance. The first two experiments will test the ability of motors to center themselves on the artificial spindle and reorganize the spindle microtubules. The third experiment will test the ability of a C-terminal kinesin motor to focus the microtubule ends into the tight clusters found in cells. By recreating the complex intracellular architecture of the mitotic spindle in vitro, this approach will uncover the nanoscale dynamic interactions of motor proteins and microtubules underlying mitosis in a way that is not possible using current single-molecule measurements or measurements in intact cells. Relevance: The goal of this work is to develop a new approach to studying the molecular mechanisms underlying cell division in animal cells. Because cancer results from uncontrolled cell division, this work is an important step towards developing novel anti-tumor therapies.